Vector Integration

A major advantage of retroviral vectors has been that they integrate into the host cell genome. The only other vector system that allows efficient integration is based on adeno-associated virus, reviewed elsewhere in this book. A great deal is known about the process of retroviral integration, which is carried out by the viral integrase protein. Integrase recognizes sequences at the ends of the LTRs of the DNA provirus (the att sites, Fig. 2), and inserts the provirus more or less randomly

Figure 3 Basic retroviral vector design. The packaging construct provides all the viral proteins in trans to the vector genome, which codes for no viral proteins but retains all the necessary cis elements. The deletion of the packaging signal from the packaging construct prevents its incorporation into viral particles.

Figure 3 Basic retroviral vector design. The packaging construct provides all the viral proteins in trans to the vector genome, which codes for no viral proteins but retains all the necessary cis elements. The deletion of the packaging signal from the packaging construct prevents its incorporation into viral particles.

into the host genome, although some sequence preferences have been reported for human immunodeficiency virus-1 (HIV-1) and its vector derivatives (20,21).

The ability of vectors to integrate is a two-edged sword. On the one hand it allows for the possibility of stable long-term gene expression, with the integrated provirus being passed on to all daughter cells. On the other hand, the possibility of insertion into a nonfavorable site also exists, which could influence both the ability of the vector to drive gene expression, and also interfere with the normal functioning of nearby host genes. Most worryingly, the vector insertion event could lead to the activation of an oncogene or inactivation of a tumor suppressor gene, with the risk of subsequent tumor formation. Indeed retroviruses were first identified on the basis of their ability to cause oncogenic transformation, and the possibility of insertional mutagenesis is of great concern. This is discussed in greater detail in Section VII.B.

One possible way around the negative aspects of retroviral integration would be to engineer the integrase protein to direct integration only into certain preselected regions of the host cell genome. The rationale for such an approach is based on the integration site preference exhibited by the related integrase protein of certain yeast transposable elements, such as Ty3, which preferentially integrates upstream of Pol III promoters (22). To date, chimeras between the integrase proteins of Ty3 and MuLV have been shown to be functional, although no redirection of MuLV integration has yet been demonstrated (23).

In a different approach, site-specific integration is being attempted through engineering of the retroviral integrase protein to contain additional DNA targeting domains that will direct the integration complex to specific sites. Some specificity of site selection has been demonstrated in vitro for chimeras containing DNA-binding zinc finger proteins (24), but the ability to redirect integration in vivo has not yet been demonstrated (reviewed in 25).

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